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Detection and Characterization of a Nicotinic Acid-Replacing Substance in Vitamin-Free Acid Hydrolyzed Casein
Detection and Characterization of a Nicotinic Acid-Replacing Substance in Vitamin-Free Acid Hydrolyzed Casein T. J. K E R R , M. V. W I L L I A M S , 1 J. J. ROWE, 2 G. J. T R I T Z , 1 and C. J. W A S H A M 3 Department of Microbiology University of Georgia Athens 30602
ABSTRACT
Auxotrophs of Escbericbia coli defective in biosynthesis of nicotinamide adenine dinucleotide are able to proliferate in a minimal medium containing either nicotinic acid or vitamin-free acid hydrolyzed casein (casamino acids). The active component in acid-hydrolyzed casein is not nicotinic acid or nicotinamide as determined by partition chromatography, analysis for teritary and quarternary amines, and the Mueller and Fox reaction for nicotinic acid. tt is not affected by proteinases. The activity of acid-hydrolyzed casein is lost by acid or alkaline hydrolysis, ashing, and charcoal filtration. The active factor has a molecular weight of less than 500 and is slightly soluble in n-butanol. It is insoluble in ether and chloroform. INTRODUCTION
The pathway for biosynthesis of nicotinamide adenine dinucleotide (NAD) in Escbericbia coli is only partially defined. Aspartic acid and dihydroxyacetone phosphate condense to form eventually quinolinic acid (QA). The number of enzymatic steps in this process is not definitely known. However, two genetic loci have been mapped that code for enzymes in this process (13). Quinolinic acid is converted to nicotinic acid mononucleotide, which gives rise to nicotinic acid adenine dinucleotide and
then to NAD. The NAD is cycled via the pyridine nucleotide cycle through nicotinamide, nicotinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide back to NAD (4). Escbericbia coti will preferentially take up exogenous nicotinic acid, nicotinamide, or both, rather than synthesize NAD de novo from aspartate. The vitamin is introduced into the pyridine nucleotide cycle and converted to NAD. We reported that a medium of minimal basal salts supplemented with vitamin-free casamino acids (CAA) supported the growth of had auxotrophs of E. coli (5). The effect was equally noticeable with mutants defective at the nadA, nadB, nadC, or nadR locus. The commercial production of vitamin-free casamino acids is accomplished by acid hydrolysis of casein, the principal protein of milk. This hydrolyzed product is passed through charcoal filters to remove vitamins and then is sold as a "vitamin-free" product for supplementation of various bacteriological media. This product is used in media for the biological assay of nicotinic acid (niacin). The standard assay involves measurement of growth of Lactobacittus arabinosus 17-5 in this medium in response to addition of known quantities of nicotinic acid or nicotinamide (12). In this paper we quantirate and characterize partially a compound in casamino acids that can replace nicotinic acid as a growth factor for nad mutants of E. coli. MATERIALS AND METHODS Organisms and Culture Media
Received May 3, 1982. 1Department of Microbiology and Immunology, Kirksville College of Osteopathic Medicine, Kirksville, MO 63501. ~Department of Biology, University of Dayton, Dayton, OH 45469. 3VITEX/R & H Division, Mallinckrodt, Inc., St. Louis, MO 63134. 1983 J Dairy Sci. 66:743-749
743
Both organisms were derivatives of E. coli K-12. Strain UTH 4460 is a nadA mutant; strain UTH 4150 harbors a nadB mutation. The minimal medium contained the following ingredients per liter: K2HPO, 1 g; KH2PO4, 3 g; sodium citrate, .5 g; (NH4)2SO4, 1.0 g; MgSO4"7H20, 11 g; thiamin, 1.0 mg; and glu-
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KERR ET AL.
cose, 2.0 g. The pH was adjusted to 8.0 with 2N HC1. The vitamin-free acid hydrolysate of casein was commercially available CAA purified from Difco Laboratories, Inc., Detroit, MI. Biological Assay of Active Component
Optimum pH for utilization of the nicotinic acid-replacing activity in acid-hydrolyzed casein was 8.0. Therefore, all growth experiments were at pH 8.0. In a typical experiment, the inoculum (either a nadA or nadB mutant of E. coti) was grown overnight in a minimal medium containing 10 .4 M nicotinic acid. Five milliliters of this culture were centrifuged at 6000 x g for 15 min in a Sorval Model DE Centrifuge. The supernatant fluid was decanted, and 5 ml sterile .8% saline solution were added to the pellet. Cells were resuspended with an SM vortex mixer. This procedure was repeated twice, and the suspended cells were diluted to 10 -s of the original concentration. Six milliliters of minimal medium containing .5 t~l of the material to be tested were inoculated with .1 ml of diluted cells. The cultures were incubated for 12 to 14 h at 3 7 ° C i n a N e w Brunswick reciprocal-shaking incubator. All cultures that grew were checked for revertants by inoculation of minimal agar plates and incubation of these plates for 36 h at 37°C. Separation Techniques
Uhrafihration of acid-hydrolyzed casein was by an Amicon Micro-Ultrafiltration System Model 8 MC with a U-500 membrane. One dimensional descending partition chromatography was with Whatman Number 1 paper. Solvent systems are described in the text. All separations were at ambient temperature for 15 h in an enclosed equilibrated chamber. Amino Acid Analysis
A .4% aqueous solution of acid hydrolyzed casein was prepared and diluted 1:4 with sodium citrate buffer (2N Na +, pH 2.2) for amino acid analysis. The analysis was with a Beckman Model 121 amino acid analyzer and a 10 h physiological program. The basic amino acids were separated with a Beckman Resin PA35 (25 cm × .9 cm) column and the acidic acid and neutral amino acids with a PA-28 (50 cm × Journal of Dairy Science Vol. 66, No. 4, 1983
.9 cm) column. Sodium citrate buffers were used at flow rates of 50 ml/h. Chemical Assays
Analyses of samples for tertiary ring nitrogen were according to the method of Konig (8). Quarternary ring nitrogen was determined by the method of Kodicek and Reddi (7). Nicotinic acid was assayed by Mueller and Fox reaction (9). Chemicals
Nicotinic acid, nicotinamide, amino acids, and pyridine nucleotides were purchased from Sigma Chemical Company. Activated charcoal (Norit-A), acetic acid, and all organic solvents were obtained from Fisher Chemical Company. All other chemicals were of the highest quality commercially available and were obtained from major supply houses. Sephadex G-10 and G-15 and chromatographic column were from Pharmacia Fine Chemicals. Bioassay
Paper chromatograms were assayed biologically by 1-cm wide strips excised from the chromatograms. Each strip was placed on the surface of a minimal agar plate which had been spread with a lawn of a had mutant. These plates were incubated upright at 37°C for up to 72 h. Biological activity was indicated by a radius of growth on the surface of the plate extending outward from the paper strip.
RESULTS AND DISCUSSION
Treatment of acid-hydrolyzed casein with trypsin, pepsin, or a commercial mixture of proteolytic enzymes caused no loss of biological activity. Therefore, it can be concluded that either no peptide bonds were available to the enzymes or that the breakage of the peptide bond(s) in the molecule had no effect on its activity. Autoclaving of a 10% solution of acidhydrolyzed casein for 15 rain at 138 kPa had no effect on the activity. However, acid or alkaline hydrolysis for 12 h in the autoclave at 138 kPa destroyed the activity. It appeared that the active principal was organic with hydrolyzable bonds. The organic nature of the compound was confirmed by the observation that ashing
CASEIN HYDROLYSATE of the acid-hydrolyzed casein resulted in complete loss of, activity. An aqueous solution of acid-hydrolyzed casein was subjected to ultrafiltration analysis to approximate its molecular weight. The compound passed through an anisotropic membrane (pore diameter of .1 nm) with a macrosolute retention of 500 MW, indicating that the active compound has < 500 MW. In an attempt to extract the active principal from acid-hydrolyzed casein, its solubility in various common solvents was determined. The compound was not soluble in absolute ethanol, acetone, chloroform, phenol, or ether. The only solvent tested that showed any ability to extract the compound was n-butanol, in which there was slight solubility. Filtration of an aqueous solution of acidhydrolyzed casein through activated charcoal resulted in the loss of most of the activity of the solution. It was assumed that the active fraction was absorbed on the charcoal; however, the activity could not be eluted. Control experiments indicated that both nicotinic acid and nicotinamide could be eluted partially from charcoal (Table 1).
Comparison of Active Component with Pyridine Nucleotide Cycle Intermediates
Although acid-hydrolyzed casein is sold as CAA and is said to be free of vitamins, several reports have been published indicating the presence of trace amounts of pyridine vitamins (i, 11). These amounts are generally about onetenth of the minimal amount required for growth of E. coll. To rule out the possibility that stimulation of E. coli was due to the presence of the known immediate precursors of NAD, we compared biologically and chemically acid-hydrolyzed casein with common biological pyridines. All pyridines either quench ultraviolet (UV) light or fluoresce in its presence. When a solution of acid-hydrolyzed casein was subjected to one-dimensional descending partition paper chromatography utilizing an ethanolammonium hydroxide solvent system, only one fluorescent spot and no quenching spots were detected on the chromatogram (Figure 1). The Rf of this spot did not correspond to that of any of the standards, and the spot did not have biological activity. Also, all of the standards
745
TABLE 1. Effects of analysis of various treatments upon the biological activity of vitamin-free acidhydrolyzed casein (casamino acids). Treatment
Effect
Charcoal clarification Exposure to pepsin Exposure to trypsin Exposure to protease Ultrafiltration
Activity lost No effect No effect No effect Activity passes through 500 MW filter No effect No effect No effect No effect No effect Partial extraction Activity lost No effect Activity lost Activity lost Activity lost
Ether extraction Ethanol extraction Acetone extraction Chloroform extraction Phenol extraction Butanol extraction Ashing Autoclaving HC1 hydrolysis H2SO4 hydrolysis Ba(OH)2 hydrolysis
quenched but did not fluoresce under UV light. The nicotinic acid-substituting compound of the acid-hydrolyzed casein migrated to a position comparable to that of nicotinic acid and nicotinamide, although no UV reactive spot was detected at this location. The partition of acid-hydrolyzed casein in a
CAA
NA
NAD
QA
|AA
.1 --I
--I
--n
--I
A.
j, -
3-
7¥
EZ; I
Figure 1. One-dimension descending partition chromatography of vitamin-free casamino acids (CAA) and various biological pyridines in a 95% ethanol 58% ammonium hydroxide (95:5) solvent. - , lack of support of growth of nad mutant; +, support of growth of nad mutant; ~[[~, quenching of UV light; ~, fluorescence under UV light. Journal of Dairy Science Vol. 66, No. 4, 1983
746
KERR ET AL. CAA
MAD
NA
CtA
NA•
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.
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Figure 2. One-dimension descending partition chromatography of vitamin-free casamino acids (CAA) and various biological pyridines in 1-butanol-acetic acid-water (250:60:250) solvent. - , lack of support of growth of nad mutant; +, support of growth o f n a d mutant; ~ 7 , quenching of UV light; ~I~P, fluorescence under UV light.
descending paper chromatography system utilizing a butanol-acetic acid-water solvent is depicted in Figure 2. In this case, biological activity of the acid-hydrolyzed casein remained at the origin. Again, no UV quenching or fluorescent spot was detected at the biologically-active area. Both N A D and QA preparations appear to be contaminated; positions of NAD and QA at the origin were inferred by the fact that this was the position of the greatest amount of quenching. Furthermore, reports indicate that these compounds do not migrate in this solvent system (2). Thus, the active component of the acid-hydrolyzed casein did not migrate with any of the standards when both solvent systems were considered. Also, the active component did not quench UV light or fluoresce in its presence. This system is capable of detecting as little as 10 -9 moles of a pyridine if concentrated in a spot 1 cm or less in diameter. To confirm that the active factor of acidhydrolyzed casein differed from known pyridine nucleotide c y c l e intermediates, a series of chemical analyses were performed on acidhydrolyzed casein and a variety of standard compounds. The Konig reaction (8) is a test for a tertiary ring nitrogen. This test may be with either p-aminobenzoic or benzidine; each reagent yields a different color in a positive test. Sensitivities of the test compounds differ Journal of Dairy Science Vol. 66, No. 4, 1983
in each test and with each reactant. A positive test, in the reaction utilizing p-aminobenzoic acid, is indicated by an orange color in visible light. This color may or may not vary when viewed under UV light. Either a) there was no compound in acid-hydrolyzed casein with a tertiary ring nitrogen, b) the amount of the compound containing the ring nitrogen was below the limits of resolution of the test, or c) compounds in the acid-hydrolyzed casein interfered with the' test. In the reaction employing benzidine, a tertiary ring nitrogen is indicated by development of a red-orangebrown color. Again, the acid-hydrolyzed casein gave a negative reaction. The Kodicek and Reddi reaction (7) for a quarternary ring nitrogen relies upon development of a blue fluorescence under UV light. The sample CAA failed to yield a positive test. The Mueller and F o x reaction (9) is a colorimetric test specific for nicotinic acid. The standard curve indicated that as little as 1 #tg nicotonic acid/ml was detected by this assay. A 10% (wt/vol) solution of acid-hydrolyzed casein failed to yield a positive reaction for nicotinic acid. Determination of Minimal Amount of Nicotinic Acid Required for Growth
To quantify the active factor of acidhydrolyzed casein relative to nicotinic acid, a biological assay used both n a d A and n a d B dichotomistic mutants. This assay was not performed in the usual way (i.e., absorbance of cell protein or cell nitrogen measured at the stationary phase of growth) because preliminary experiments indicated alteration of shape of the growth curve in response to variations in concentration of acid-hydrolyzed casein. Instead, absorbance of a culture was monitored over the entire growth cycle. The n a d A mutant exhibited a normal sigmoidal growth curve when grown in a minimal broth medium supplemented with 100 ng nicotinic acid/ml (Figure 3). Reduction of nicotinic acid to 10 ng/ml resulted in a 36-h lag phase. A minimal medium supplement with 1 ng of nicotinic acid/ml did not support growth of the n a d A mutant. The growth of this same n a d A mutant in a minimal medium supplemented with various concentrations of acid-hydrolyzed casein is depicted in Figure 4. A medium supplemented
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Figure 3. Effect of nicotinic acid on the growth of an nadA mutant of E. coll.
with less than .5% acid-hydrolyzed casein (wt/ vol) did not support growth. The culture exhibited a biphasic growth curve when the medium was supplemented with acid-hydrolyzed casein at a concentration between .5% and 4%. Higher concentrations of acid-hydrolyzed casein resulted in a normal single-phase growth curve. A nadB m u t a n t exhibited similar growth responses to acid-hydrolyzed casein and nicotinic acid. The minimal amount of acidhydrolyzed casein required to achieve a normal sigmoidal growth curve and maximal absorbance at the stationary phase was 4% for the nadA mutant. This suggests that there may be two compounds in acid-hydrolyzed casein that can support growth of nicotinic acid-requiring strains.
Analysis of Composition of Acid-Hydrolyzed Casein
The possibility existed that the growthstimulating ability of acid-hydrolyzed casein was due to an amino acid or combination of amino acids. An amino acid quantitative analysis was performed on a .4% solution of Difco Casamino Acids. Concentration of each compound is in Table 2. All of the common amino
Figure 4. Effect of vitamin-free casamino acids (CAA) on the growth of an nadA m u t a n t of E. coll.
acids were present with the exception of tryptophan, which was destroyed by acid hydrolysis. Cysteine was in only trace amounts. In addition, there were 12 small unidentified ninhydrin-positive peaks. These may represen small peptides or degradation products of common amino acids. A synthetic acid-hydrolyzed casein medium was prepared that approximated the composition of the commercial product relative to amino acid, mineral, and fatty acid concentrations. This medium did not support the growth of nadA or nadB mutants of E. coli. There are reports that peptides in casein digests have growth-promoting effects (3, 6, 10). Therefore, a number of di- and tripeptides (Table 3) were tested for stimulation of growth of nad mutants, but no such stimulation was found. This study demonstrated that commercially available acid-hydrolyzed casein contains at least one compound other than nicotinic acid that will support growth of nicotinic acidrequiring mutants of E. coll. The compound is not an amino acid or a dipeptide. There is a possibility that the nicotinic acid-replacing substance may represent a previously u n k n o w n intermediate of the NAD pathway. Journal of Dairy Science Vol. 66, No. 4, 1983
748
KERR ET AL.
T A B L E 2. Percentages of ninhydrin positive compounds in vitamin-free acid hydrolyzed casein (casamino acids, Difco lot number 570131).
Compound
#moles/ ml*
Compound
Phosphoserine Aspartic acid Threo nine Serine Proline Glutamic acid Glycine Alanine Cysteine Valine Cystathionine
.075 .926 .725 1.041 2.067 3.002 .505 .766 Trace .992 .020
Methionine Isoleucine Leu cine Tyrosine Phenylalanine Ornithine Ammonia Lysine Histidine Arginine 12 small unidentified peaks
#moles/ ml .473 .659 1.512 .124 .502 Trace .299 1.280 .339 .408
*Of a .4% solution.
TABLE 3. A list of peptides that failed to exhibit biological activity in stimulating the growth of nad mutants of Escbericbia coil
L-Alanyl-L-alanine L-Alanylglycine L-Alanylgiycylglycine L-Alanyl-L-leucine L-Alanyl-L-p henylalanine L-Alanyl-L-serine L-Alanyl-tyrosine L-Arginyl-L-aspartic acid L-Methionyl-L-alanyl- L-serine L-Methionyl-L-methionine L-Methionyl-L-serine L-Histyl-L-alanine
ACKNOWLEDGMENTS
A special n o t e of t h a n k s goes to Douglas Hale of M e m o r i a l University, St. J o h n s , Newf o u n d l a n d , for his help in a n a l y z i n g t h e vitamin-free a c i d - h y d r o l y z e d casein. We also t h a n k N a n c y B o n d for e x p e r t t e c h nical assistance. This w o r k was f u n d e d b y research g r a n t P C M - 7 6 - 2 4 5 1 6 f r o m the N a t i o n a l Science F o u n d a t i o n . REFERENCES
1 Association of Vitamin Chemists. 1951. Pages 169-195 in Methods of vitamin assay, lntersci. Publ., New York, NY. 2 Averett, D. R., and G. J. Tirtz. 1976. A method for the rapid separation and characterization of Journal of Dairy Science Vol. 66, No. 4, 1983
L-Histyl-L-serine L-Histyl-L-tyrosine L-Leucyl-L-alanine L-Leucyl-L-methionine L-Leucyl-L-serine L-Lysyl-L-aspartic acid L-Lysyl-L-leucine Glycyl-L-methionine Glycyl- L-alanine-L-alanine Glycyl-L-aspartic acid D L-Alanyl-D L-leucylglycine
biological pyridines. J. Chromatogr. Sci. 14:350. 3 Demain, A. L., and D. Hendlin. 1958. Growth stimulation of a strain of Bacillus subtilis by glycine peptides. J. Bacteriol. 75:46. 4 Gholson, R. K. 1970. The pyridine nucleotide cycle. Nature (London) 212:934. 5 Kerr, T. J., and G. J. Tritz. 1973. Cross-feeding of t')scbericbia coli mutants defective in the biosynthesis of nicotinamide adenine dinucleotide. J. Bacteriol. 115:982. 6 Kahara, H., and E. E. Snell. 1952. Peptides and bacterial growth. II. L-alanine peptides and growth of Lacwbacillus casei. J. Biol. Chem. 197:791. 7 Kodicek, E., and K. K. Reddi. 1951. Paper chromatography of nicotinic acid derivatives. Nature 168: 475. 8 Konig, W. 1904. Untersuchungen sus dem organischen Laboratorium der Technischen Hochschule zu Dresden. LXIX. Uber eine neue, vom Pyridin
CASEIN H Y D R O L Y S A T E derivierende Klasse yon Farbstaffen. J. Prakt. Chem. 69:105. 9 Mueller, A., and S. H. Fox. 1946. Chemical determination of niacin. J. Biol. Chem. 167:291. 10 Prescott, J. M., V. J. Peters, and E. E. Snell. 1953. Peptides and bacterial growth. V. Serine peptides and growth of Lactobacillus delbrueckii 9649. J. Biol. Chem. 202:533. 11 Schubert, R.H.W., and G. Schleidt. 1975. Investigations on the standardization of polytrophic substances used in microbiological test procedures. I.
749
Suitability of vitamin-free casein hydrolysates as a standardizable source of N and C for bacterial growth. Abl. Bakt. Hyg., I. Abstr. Orig. B160: 173. 12 Snell, E. E., and L. D. Wright. 1941. A microbiological m e t h o d for the determination of nicotinic acid. J. Biol. Chem. 139:675. 13 Tritz, G. J., T. S. Matney, and R. K. Gholson. 1970. Mapping of the nadB locus adjacent to a previously undescribed purine locus in Escbericbia coli K-12. J. Bacteriol. 102:377.
Journal of Dairy Science Vol. 66, No. 4, 1983
ABSTRACT
Auxotrophs of Escbericbia coli defective in biosynthesis of nicotinamide adenine dinucleotide are able to proliferate in a minimal medium containing either nicotinic acid or vitamin-free acid hydrolyzed casein (casamino acids). The active component in acid-hydrolyzed casein is not nicotinic acid or nicotinamide as determined by partition chromatography, analysis for teritary and quarternary amines, and the Mueller and Fox reaction for nicotinic acid. tt is not affected by proteinases. The activity of acid-hydrolyzed casein is lost by acid or alkaline hydrolysis, ashing, and charcoal filtration. The active factor has a molecular weight of less than 500 and is slightly soluble in n-butanol. It is insoluble in ether and chloroform. INTRODUCTION
The pathway for biosynthesis of nicotinamide adenine dinucleotide (NAD) in Escbericbia coli is only partially defined. Aspartic acid and dihydroxyacetone phosphate condense to form eventually quinolinic acid (QA). The number of enzymatic steps in this process is not definitely known. However, two genetic loci have been mapped that code for enzymes in this process (13). Quinolinic acid is converted to nicotinic acid mononucleotide, which gives rise to nicotinic acid adenine dinucleotide and
then to NAD. The NAD is cycled via the pyridine nucleotide cycle through nicotinamide, nicotinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide back to NAD (4). Escbericbia coti will preferentially take up exogenous nicotinic acid, nicotinamide, or both, rather than synthesize NAD de novo from aspartate. The vitamin is introduced into the pyridine nucleotide cycle and converted to NAD. We reported that a medium of minimal basal salts supplemented with vitamin-free casamino acids (CAA) supported the growth of had auxotrophs of E. coli (5). The effect was equally noticeable with mutants defective at the nadA, nadB, nadC, or nadR locus. The commercial production of vitamin-free casamino acids is accomplished by acid hydrolysis of casein, the principal protein of milk. This hydrolyzed product is passed through charcoal filters to remove vitamins and then is sold as a "vitamin-free" product for supplementation of various bacteriological media. This product is used in media for the biological assay of nicotinic acid (niacin). The standard assay involves measurement of growth of Lactobacittus arabinosus 17-5 in this medium in response to addition of known quantities of nicotinic acid or nicotinamide (12). In this paper we quantirate and characterize partially a compound in casamino acids that can replace nicotinic acid as a growth factor for nad mutants of E. coli. MATERIALS AND METHODS Organisms and Culture Media
Received May 3, 1982. 1Department of Microbiology and Immunology, Kirksville College of Osteopathic Medicine, Kirksville, MO 63501. ~Department of Biology, University of Dayton, Dayton, OH 45469. 3VITEX/R & H Division, Mallinckrodt, Inc., St. Louis, MO 63134. 1983 J Dairy Sci. 66:743-749
743
Both organisms were derivatives of E. coli K-12. Strain UTH 4460 is a nadA mutant; strain UTH 4150 harbors a nadB mutation. The minimal medium contained the following ingredients per liter: K2HPO, 1 g; KH2PO4, 3 g; sodium citrate, .5 g; (NH4)2SO4, 1.0 g; MgSO4"7H20, 11 g; thiamin, 1.0 mg; and glu-
744
KERR ET AL.
cose, 2.0 g. The pH was adjusted to 8.0 with 2N HC1. The vitamin-free acid hydrolysate of casein was commercially available CAA purified from Difco Laboratories, Inc., Detroit, MI. Biological Assay of Active Component
Optimum pH for utilization of the nicotinic acid-replacing activity in acid-hydrolyzed casein was 8.0. Therefore, all growth experiments were at pH 8.0. In a typical experiment, the inoculum (either a nadA or nadB mutant of E. coti) was grown overnight in a minimal medium containing 10 .4 M nicotinic acid. Five milliliters of this culture were centrifuged at 6000 x g for 15 min in a Sorval Model DE Centrifuge. The supernatant fluid was decanted, and 5 ml sterile .8% saline solution were added to the pellet. Cells were resuspended with an SM vortex mixer. This procedure was repeated twice, and the suspended cells were diluted to 10 -s of the original concentration. Six milliliters of minimal medium containing .5 t~l of the material to be tested were inoculated with .1 ml of diluted cells. The cultures were incubated for 12 to 14 h at 3 7 ° C i n a N e w Brunswick reciprocal-shaking incubator. All cultures that grew were checked for revertants by inoculation of minimal agar plates and incubation of these plates for 36 h at 37°C. Separation Techniques
Uhrafihration of acid-hydrolyzed casein was by an Amicon Micro-Ultrafiltration System Model 8 MC with a U-500 membrane. One dimensional descending partition chromatography was with Whatman Number 1 paper. Solvent systems are described in the text. All separations were at ambient temperature for 15 h in an enclosed equilibrated chamber. Amino Acid Analysis
A .4% aqueous solution of acid hydrolyzed casein was prepared and diluted 1:4 with sodium citrate buffer (2N Na +, pH 2.2) for amino acid analysis. The analysis was with a Beckman Model 121 amino acid analyzer and a 10 h physiological program. The basic amino acids were separated with a Beckman Resin PA35 (25 cm × .9 cm) column and the acidic acid and neutral amino acids with a PA-28 (50 cm × Journal of Dairy Science Vol. 66, No. 4, 1983
.9 cm) column. Sodium citrate buffers were used at flow rates of 50 ml/h. Chemical Assays
Analyses of samples for tertiary ring nitrogen were according to the method of Konig (8). Quarternary ring nitrogen was determined by the method of Kodicek and Reddi (7). Nicotinic acid was assayed by Mueller and Fox reaction (9). Chemicals
Nicotinic acid, nicotinamide, amino acids, and pyridine nucleotides were purchased from Sigma Chemical Company. Activated charcoal (Norit-A), acetic acid, and all organic solvents were obtained from Fisher Chemical Company. All other chemicals were of the highest quality commercially available and were obtained from major supply houses. Sephadex G-10 and G-15 and chromatographic column were from Pharmacia Fine Chemicals. Bioassay
Paper chromatograms were assayed biologically by 1-cm wide strips excised from the chromatograms. Each strip was placed on the surface of a minimal agar plate which had been spread with a lawn of a had mutant. These plates were incubated upright at 37°C for up to 72 h. Biological activity was indicated by a radius of growth on the surface of the plate extending outward from the paper strip.
RESULTS AND DISCUSSION
Treatment of acid-hydrolyzed casein with trypsin, pepsin, or a commercial mixture of proteolytic enzymes caused no loss of biological activity. Therefore, it can be concluded that either no peptide bonds were available to the enzymes or that the breakage of the peptide bond(s) in the molecule had no effect on its activity. Autoclaving of a 10% solution of acidhydrolyzed casein for 15 rain at 138 kPa had no effect on the activity. However, acid or alkaline hydrolysis for 12 h in the autoclave at 138 kPa destroyed the activity. It appeared that the active principal was organic with hydrolyzable bonds. The organic nature of the compound was confirmed by the observation that ashing
CASEIN HYDROLYSATE of the acid-hydrolyzed casein resulted in complete loss of, activity. An aqueous solution of acid-hydrolyzed casein was subjected to ultrafiltration analysis to approximate its molecular weight. The compound passed through an anisotropic membrane (pore diameter of .1 nm) with a macrosolute retention of 500 MW, indicating that the active compound has < 500 MW. In an attempt to extract the active principal from acid-hydrolyzed casein, its solubility in various common solvents was determined. The compound was not soluble in absolute ethanol, acetone, chloroform, phenol, or ether. The only solvent tested that showed any ability to extract the compound was n-butanol, in which there was slight solubility. Filtration of an aqueous solution of acidhydrolyzed casein through activated charcoal resulted in the loss of most of the activity of the solution. It was assumed that the active fraction was absorbed on the charcoal; however, the activity could not be eluted. Control experiments indicated that both nicotinic acid and nicotinamide could be eluted partially from charcoal (Table 1).
Comparison of Active Component with Pyridine Nucleotide Cycle Intermediates
Although acid-hydrolyzed casein is sold as CAA and is said to be free of vitamins, several reports have been published indicating the presence of trace amounts of pyridine vitamins (i, 11). These amounts are generally about onetenth of the minimal amount required for growth of E. coll. To rule out the possibility that stimulation of E. coli was due to the presence of the known immediate precursors of NAD, we compared biologically and chemically acid-hydrolyzed casein with common biological pyridines. All pyridines either quench ultraviolet (UV) light or fluoresce in its presence. When a solution of acid-hydrolyzed casein was subjected to one-dimensional descending partition paper chromatography utilizing an ethanolammonium hydroxide solvent system, only one fluorescent spot and no quenching spots were detected on the chromatogram (Figure 1). The Rf of this spot did not correspond to that of any of the standards, and the spot did not have biological activity. Also, all of the standards
745
TABLE 1. Effects of analysis of various treatments upon the biological activity of vitamin-free acidhydrolyzed casein (casamino acids). Treatment
Effect
Charcoal clarification Exposure to pepsin Exposure to trypsin Exposure to protease Ultrafiltration
Activity lost No effect No effect No effect Activity passes through 500 MW filter No effect No effect No effect No effect No effect Partial extraction Activity lost No effect Activity lost Activity lost Activity lost
Ether extraction Ethanol extraction Acetone extraction Chloroform extraction Phenol extraction Butanol extraction Ashing Autoclaving HC1 hydrolysis H2SO4 hydrolysis Ba(OH)2 hydrolysis
quenched but did not fluoresce under UV light. The nicotinic acid-substituting compound of the acid-hydrolyzed casein migrated to a position comparable to that of nicotinic acid and nicotinamide, although no UV reactive spot was detected at this location. The partition of acid-hydrolyzed casein in a
CAA
NA
NAD
QA
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Figure 1. One-dimension descending partition chromatography of vitamin-free casamino acids (CAA) and various biological pyridines in a 95% ethanol 58% ammonium hydroxide (95:5) solvent. - , lack of support of growth of nad mutant; +, support of growth of nad mutant; ~[[~, quenching of UV light; ~, fluorescence under UV light. Journal of Dairy Science Vol. 66, No. 4, 1983
746
KERR ET AL. CAA
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Figure 2. One-dimension descending partition chromatography of vitamin-free casamino acids (CAA) and various biological pyridines in 1-butanol-acetic acid-water (250:60:250) solvent. - , lack of support of growth of nad mutant; +, support of growth o f n a d mutant; ~ 7 , quenching of UV light; ~I~P, fluorescence under UV light.
descending paper chromatography system utilizing a butanol-acetic acid-water solvent is depicted in Figure 2. In this case, biological activity of the acid-hydrolyzed casein remained at the origin. Again, no UV quenching or fluorescent spot was detected at the biologically-active area. Both N A D and QA preparations appear to be contaminated; positions of NAD and QA at the origin were inferred by the fact that this was the position of the greatest amount of quenching. Furthermore, reports indicate that these compounds do not migrate in this solvent system (2). Thus, the active component of the acid-hydrolyzed casein did not migrate with any of the standards when both solvent systems were considered. Also, the active component did not quench UV light or fluoresce in its presence. This system is capable of detecting as little as 10 -9 moles of a pyridine if concentrated in a spot 1 cm or less in diameter. To confirm that the active factor of acidhydrolyzed casein differed from known pyridine nucleotide c y c l e intermediates, a series of chemical analyses were performed on acidhydrolyzed casein and a variety of standard compounds. The Konig reaction (8) is a test for a tertiary ring nitrogen. This test may be with either p-aminobenzoic or benzidine; each reagent yields a different color in a positive test. Sensitivities of the test compounds differ Journal of Dairy Science Vol. 66, No. 4, 1983
in each test and with each reactant. A positive test, in the reaction utilizing p-aminobenzoic acid, is indicated by an orange color in visible light. This color may or may not vary when viewed under UV light. Either a) there was no compound in acid-hydrolyzed casein with a tertiary ring nitrogen, b) the amount of the compound containing the ring nitrogen was below the limits of resolution of the test, or c) compounds in the acid-hydrolyzed casein interfered with the' test. In the reaction employing benzidine, a tertiary ring nitrogen is indicated by development of a red-orangebrown color. Again, the acid-hydrolyzed casein gave a negative reaction. The Kodicek and Reddi reaction (7) for a quarternary ring nitrogen relies upon development of a blue fluorescence under UV light. The sample CAA failed to yield a positive test. The Mueller and F o x reaction (9) is a colorimetric test specific for nicotinic acid. The standard curve indicated that as little as 1 #tg nicotonic acid/ml was detected by this assay. A 10% (wt/vol) solution of acid-hydrolyzed casein failed to yield a positive reaction for nicotinic acid. Determination of Minimal Amount of Nicotinic Acid Required for Growth
To quantify the active factor of acidhydrolyzed casein relative to nicotinic acid, a biological assay used both n a d A and n a d B dichotomistic mutants. This assay was not performed in the usual way (i.e., absorbance of cell protein or cell nitrogen measured at the stationary phase of growth) because preliminary experiments indicated alteration of shape of the growth curve in response to variations in concentration of acid-hydrolyzed casein. Instead, absorbance of a culture was monitored over the entire growth cycle. The n a d A mutant exhibited a normal sigmoidal growth curve when grown in a minimal broth medium supplemented with 100 ng nicotinic acid/ml (Figure 3). Reduction of nicotinic acid to 10 ng/ml resulted in a 36-h lag phase. A minimal medium supplement with 1 ng of nicotinic acid/ml did not support growth of the n a d A mutant. The growth of this same n a d A mutant in a minimal medium supplemented with various concentrations of acid-hydrolyzed casein is depicted in Figure 4. A medium supplemented
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with less than .5% acid-hydrolyzed casein (wt/ vol) did not support growth. The culture exhibited a biphasic growth curve when the medium was supplemented with acid-hydrolyzed casein at a concentration between .5% and 4%. Higher concentrations of acid-hydrolyzed casein resulted in a normal single-phase growth curve. A nadB m u t a n t exhibited similar growth responses to acid-hydrolyzed casein and nicotinic acid. The minimal amount of acidhydrolyzed casein required to achieve a normal sigmoidal growth curve and maximal absorbance at the stationary phase was 4% for the nadA mutant. This suggests that there may be two compounds in acid-hydrolyzed casein that can support growth of nicotinic acid-requiring strains.
Analysis of Composition of Acid-Hydrolyzed Casein
The possibility existed that the growthstimulating ability of acid-hydrolyzed casein was due to an amino acid or combination of amino acids. An amino acid quantitative analysis was performed on a .4% solution of Difco Casamino Acids. Concentration of each compound is in Table 2. All of the common amino
Figure 4. Effect of vitamin-free casamino acids (CAA) on the growth of an nadA m u t a n t of E. coll.
acids were present with the exception of tryptophan, which was destroyed by acid hydrolysis. Cysteine was in only trace amounts. In addition, there were 12 small unidentified ninhydrin-positive peaks. These may represen small peptides or degradation products of common amino acids. A synthetic acid-hydrolyzed casein medium was prepared that approximated the composition of the commercial product relative to amino acid, mineral, and fatty acid concentrations. This medium did not support the growth of nadA or nadB mutants of E. coli. There are reports that peptides in casein digests have growth-promoting effects (3, 6, 10). Therefore, a number of di- and tripeptides (Table 3) were tested for stimulation of growth of nad mutants, but no such stimulation was found. This study demonstrated that commercially available acid-hydrolyzed casein contains at least one compound other than nicotinic acid that will support growth of nicotinic acidrequiring mutants of E. coll. The compound is not an amino acid or a dipeptide. There is a possibility that the nicotinic acid-replacing substance may represent a previously u n k n o w n intermediate of the NAD pathway. Journal of Dairy Science Vol. 66, No. 4, 1983
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KERR ET AL.
T A B L E 2. Percentages of ninhydrin positive compounds in vitamin-free acid hydrolyzed casein (casamino acids, Difco lot number 570131).
Compound
#moles/ ml*
Compound
Phosphoserine Aspartic acid Threo nine Serine Proline Glutamic acid Glycine Alanine Cysteine Valine Cystathionine
.075 .926 .725 1.041 2.067 3.002 .505 .766 Trace .992 .020
Methionine Isoleucine Leu cine Tyrosine Phenylalanine Ornithine Ammonia Lysine Histidine Arginine 12 small unidentified peaks
#moles/ ml .473 .659 1.512 .124 .502 Trace .299 1.280 .339 .408
*Of a .4% solution.
TABLE 3. A list of peptides that failed to exhibit biological activity in stimulating the growth of nad mutants of Escbericbia coil
L-Alanyl-L-alanine L-Alanylglycine L-Alanylgiycylglycine L-Alanyl-L-leucine L-Alanyl-L-p henylalanine L-Alanyl-L-serine L-Alanyl-tyrosine L-Arginyl-L-aspartic acid L-Methionyl-L-alanyl- L-serine L-Methionyl-L-methionine L-Methionyl-L-serine L-Histyl-L-alanine
ACKNOWLEDGMENTS
A special n o t e of t h a n k s goes to Douglas Hale of M e m o r i a l University, St. J o h n s , Newf o u n d l a n d , for his help in a n a l y z i n g t h e vitamin-free a c i d - h y d r o l y z e d casein. We also t h a n k N a n c y B o n d for e x p e r t t e c h nical assistance. This w o r k was f u n d e d b y research g r a n t P C M - 7 6 - 2 4 5 1 6 f r o m the N a t i o n a l Science F o u n d a t i o n . REFERENCES
1 Association of Vitamin Chemists. 1951. Pages 169-195 in Methods of vitamin assay, lntersci. Publ., New York, NY. 2 Averett, D. R., and G. J. Tirtz. 1976. A method for the rapid separation and characterization of Journal of Dairy Science Vol. 66, No. 4, 1983
L-Histyl-L-serine L-Histyl-L-tyrosine L-Leucyl-L-alanine L-Leucyl-L-methionine L-Leucyl-L-serine L-Lysyl-L-aspartic acid L-Lysyl-L-leucine Glycyl-L-methionine Glycyl- L-alanine-L-alanine Glycyl-L-aspartic acid D L-Alanyl-D L-leucylglycine
biological pyridines. J. Chromatogr. Sci. 14:350. 3 Demain, A. L., and D. Hendlin. 1958. Growth stimulation of a strain of Bacillus subtilis by glycine peptides. J. Bacteriol. 75:46. 4 Gholson, R. K. 1970. The pyridine nucleotide cycle. Nature (London) 212:934. 5 Kerr, T. J., and G. J. Tritz. 1973. Cross-feeding of t')scbericbia coli mutants defective in the biosynthesis of nicotinamide adenine dinucleotide. J. Bacteriol. 115:982. 6 Kahara, H., and E. E. Snell. 1952. Peptides and bacterial growth. II. L-alanine peptides and growth of Lacwbacillus casei. J. Biol. Chem. 197:791. 7 Kodicek, E., and K. K. Reddi. 1951. Paper chromatography of nicotinic acid derivatives. Nature 168: 475. 8 Konig, W. 1904. Untersuchungen sus dem organischen Laboratorium der Technischen Hochschule zu Dresden. LXIX. Uber eine neue, vom Pyridin
CASEIN H Y D R O L Y S A T E derivierende Klasse yon Farbstaffen. J. Prakt. Chem. 69:105. 9 Mueller, A., and S. H. Fox. 1946. Chemical determination of niacin. J. Biol. Chem. 167:291. 10 Prescott, J. M., V. J. Peters, and E. E. Snell. 1953. Peptides and bacterial growth. V. Serine peptides and growth of Lactobacillus delbrueckii 9649. J. Biol. Chem. 202:533. 11 Schubert, R.H.W., and G. Schleidt. 1975. Investigations on the standardization of polytrophic substances used in microbiological test procedures. I.
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Suitability of vitamin-free casein hydrolysates as a standardizable source of N and C for bacterial growth. Abl. Bakt. Hyg., I. Abstr. Orig. B160: 173. 12 Snell, E. E., and L. D. Wright. 1941. A microbiological m e t h o d for the determination of nicotinic acid. J. Biol. Chem. 139:675. 13 Tritz, G. J., T. S. Matney, and R. K. Gholson. 1970. Mapping of the nadB locus adjacent to a previously undescribed purine locus in Escbericbia coli K-12. J. Bacteriol. 102:377.
Journal of Dairy Science Vol. 66, No. 4, 1983